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Biochemistry 2010, 49, 752–760 DOI: 10.1021/bi901599u
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Structural and Functional Studies of Aspergillus clavatus N5-Carboxyaminoimidazole Ribonucleotide Synthetase†,‡ )
James B. Thoden,§ Hazel M. Holden,*,§ Hanumantharao Paritala, and Steven M. Firestine*, §
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 and Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, Michigan 48201 Received September 11, 2009; Revised Manuscript Received November 20, 2009
N5-Carboxyaminoimidazole ribonucleotide synthetase (N5-CAIR synthetase), a key enzyme in microbial de novo purine biosynthesis, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to N5-CAIR. To date, this enzyme has been observed only in microorganisms, and thus, it represents an ideal target for antimicrobial drug development. Here we report the cloning, crystallization, and three-dimensional structural analysis of Aspergillus clavatus N5-CAIR synthetase solved in the presence of either Mg2ATP or MgADP and AIR. These structures, determined to 2.1 and 2.0 A˚, respectively, revealed that AIR binds in a pocket analogous to that observed for other ATP-grasp enzymes involved in purine metabolism. On the basis of these models, a site-directed mutagenesis study was subsequently conducted that focused on five amino acid residues located in the active site region of the enzyme. These investigations demonstrated that Asp 153 and Lys 353 play critical roles in catalysis without affecting substrate binding. All other mutations affected substrate binding and, in some instances, catalysis as well. Taken together, the structural and kinetic data presented here suggest a catalytic mechanism whereby Mg2ATP and bicarbonate first react to form the unstable intermediate carboxyphosphate. This intermediate subsequently decarboxylates to CO2 and inorganic phosphate, and the amino group of AIR, through general base assistance by Asp 153, attacks CO2 to form N5-CAIR. ABSTRACT:
Arguably, one of the most important developments in the history of modern medicine has been the discovery of antibiotics. The golden age of antibacterial drug discovery began during the 1940s, and by the beginning of the 1970s, most of the major classes of antibiotics currently in clinical use had been discovered (1, 2). The rate of antimicrobial drug discovery has since slowed as evidenced by the fact that only two new classes of antibiotics, the lipopeptides and the oxazolidinones, have been brought to the market within the past 40 years (3, 4). Unfortunately, as the pharmaceutical industry’s interest in antibiotic drug discovery has waned, resistance to existing antibiotics has grown. Recent studies have shown that approximately 50% of all Staphylococcus aureus infections are methicillin resistant, and strikingly, S. aureus resistance to most other existing antibiotics has been detected in clinical settings (1, 5, 6). These results foreshadow the day in which bacteria become resistant to all known antibiotics. The increasing prevalence of antibiotic resistant infections has led to a recent, renewed interest in antibiotic drug development. For example, studies have examined enzymes such as pantothenate kinase (7), tRNA synthetase (8), and DNA ligase (9), or biosynthetic pathways such as those for the production of isoprenoids (10) and aromatic amino acid synthesis (11), as † This research was supported in part by National Institutes of Health Grants DK47814 to H.M.H. and GM087467 to S.M.F. ‡ X-ray coordinates have been deposited in the Research Collaboratory for Structural Bioinformatics, Rutgers University (Protein Data Bank entries 3K5H and 3K5I). *To whom correspondence should be addressed. E-mail: sfirestine@ wayne.edu or
[email protected]. Fax: (313) 577-2033. Phone: (313) 577-0455.
pubs.acs.org/Biochemistry
Published on Web 11/24/2009
potential antibacterial drug targets. One unexplored pathway for antimicrobial drug design is de novo purine biosynthesis, which is markedly different in microorganisms and humans (12-16). As revealed in studies conducted in the 1980s, bacteria, yeast, and fungi synthesize the purine intermediate, inosine monophosphate (IMP), via 11 enzymatic steps, whereas in humans, only 10 steps are required. The additional step is catalyzed by the enzyme N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, which converts 5-aminoimidazole ribonucleotide (AIR)1 to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) as indicated in Scheme 1 (12, 13, 16). This activity is not required for the synthesis of IMP in humans, and since humans have no homologues for this enzyme, N5CAIR synthetase represents an attractive target for drug development. Indeed, we have discovered a series of small, druglike inhibitors of this enzyme and have shown that these agents inhibit bacterial growth on purine deficient media (17). To further our drug discovery efforts and to enhance our understanding of N5-CAIR synthetase, we embarked on a series of structural and enzymological investigations. Recently, our laboratories reported the molecular structures of the Mg/ATP and Mg/ADP 3 Pi complexes of N5-CAIR synthetase from Escherichia coli (18). These two models provided us with a detailed view of the protein before and after ATP hydrolysis, and we were able to use this information to suggest a possible catalytic 1
Abbreviations: AIR, 5-aminoimidazole ribonucleotide; AMPPNP, adenosine 50 -(β,γ-imido)triphosphate; CAIR, 4-carboxy-5-aminoimidazole ribonucleotide; CHES, N-cyclohexyl-2-aminoethanesulfonic acid; MWC, molecular weight cutoff; Tris, tris(hydroxymethyl)aminomethane. r 2009 American Chemical Society
Article Scheme 1
Biochemistry, Vol. 49, No. 4, 2010 Table 1: X-ray Data Collection Statistics enzyme complexed with Mg2ATP
mechanism for the enzyme. Unfortunately, numerous efforts aimed at obtaining the structure of the enzyme in the presence of AIR failed. Due to the mechanistic, structural, and medicinal importance of this information, we initiated a study of N5-CAIR synthetase from Aspergillus clavatus, a common soil fungus. As observed in other eukaryotic microorganisms, the A. clavatus N5-CAIR synthetase exists as a bifunctional protein with the next enzyme in the pathway, N5-CAIR mutase (16). For this investigation, we cloned only the N5-CAIR synthetase domain, overexpressed and purified the protein, and determined its structure in the presence of either Mg2ATP or MgADP and AIR. In addition, we changed five key residues in the AIR binding pocket (Glu 73, Tyr 152, Asp 153, Arg 155, and Lys 353) via site-directed mutagenesis to probe their function within the active site of the enzyme. Taken together, these studies provide new, detailed information on those residues critical for substrate binding and for catalysis in the N5-CAIR synthetases. MATERIALS AND METHODS Cloning, Expression, and Purification. Genomic DNA from A. clavatus (ATCC 1007) was isolated by standard procedures. The fragment of the ADE2 gene encoding N5-CAIR synthetase (residues 1-383) was polymerase chain reactionamplified using primers that introduced 50 NdeI and 30 NotI sites. The purified polymerase chain reaction (PCR) product was A-tailed and ligated into a pGEM-T (Promega) vector for screening and sequencing. Subsequently, two introns present in the gene encoding N5-CAIR synthetase were excised by mutagenesis. PurK-pGEM-T vector constructs of the correct sequence were then appropriately digested and ligated into a pET28b(þ) (Novagen) plasmid for protein expression with an N-terminal His tag of the following sequence (MGSSHHHHHHSSENLYFQGH). The N5-CAIR synthetase-pET28 plasmid was used to transform HMS174(DE3) E. coli cells (Novagen). The cultures in Luria-Bertani media were grown at 37 °C with shaking until an optical density of ∼0.8 was measured at 600 nM. The cultures were then cooled to 16 °C; isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 1.0 mM, and induction was allowed to proceed for 18 h. N5-CAIR synthetase was purified by standard methods with nickel nitrilotriacetic acid resin. Purified N5-CAIR synthetase was dialyzed against 10 mM Tris-HCl and 200 mM NaCl (pH 8). Following dialysis, the sample was concentrated to ∼15 mg/mL and frozen in liquid nitrogen. To investigate the roles of Glu 73, Tyr 152, Asp 153, Arg 155, and Lys 353 in the catalytic mechanism of N5-CAIR synthetase, the following point mutations were constructed via the Stratagene QuikChange method: E73A, Y152A, D153A, R155A, and K353A. All modified genes were sequenced to verify that no other changes had been inadvertently introduced. Mutant proteins were expressed and purified in the manner described for the wild-type enzyme. Structural Analysis of N5-CAIR Synthetase. Crystallization conditions were initially surveyed by the hanging drop
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enzyme complexed with MgADP and AIR
space group P21 P21 resolution limits (A˚) 30.0-2.1 (2.2-2.1)b 30.0-2.0 (2.1-2.0)b no. of independent 104348 (12740)b 118616 (15717)b reflections completeness (%) 93.6 (86.5)b 91.9 (86.4)b redundancy 4.0 (1.8)b 2.9 (1.5)b avg I/avg σ(I) 9.6 (2.2)b 7.3 (2.0)b Rsym (%)a 8.7 (30.9)b 8.9 (28.6)b P P P a Rsym=( | I - I|/ I)100. bStatistics for the highest-resolution bin.
method of vapor diffusion with a sparse matrix screen developed in the laboratory, using the protein incubated with 10 mM ATP and 40 mM MgCl2. X-ray diffraction quality crystals were grown via the hanging drop method against 15-19% monomethylether poly(ethylene glycol) 5000 and 200 mM NaCl buffered with 100 mM CHES (pH 9.0). The crystals belonged to space group P21 with two dimers per asymmetric unit and the following unit cell dimensions: a=75.9 A˚, b=134.5 A˚, c=99.6 A˚, and β=107.0°. Native X-ray data were measured at 100 K using a Bruker AXS Platinum 135 CCD detector controlled with the Proteum software suite (Bruker AXS Inc.). The X-ray source was Cu KR radiation from a Rigaku RU200 X-ray generator equipped with Montel optics and operated at 50 kV and 90 mA. These X-ray data were processed with SAINT version 7.06A (Bruker AXS Inc.) and internally scaled with SADABS version 2005/1 (Bruker AXS Inc.). Prior to X-ray data collection at 100 K, the crystals were stabilized by being transferred into a 25% monomethylether poly(ethylene glycol) 5000 solution containing 400 mM NaCl, 10 mM ATP, 40 mM MgCl2, and 15% ethylene glycol. In an attempt to prepare a ternary complex of N5-CAIR synthetase with MgAMPPNP and AIR, crystals of the apoenzyme were grown under the same conditions described above and subsequently soaked in 10 mM AMPPNP, 40 mM MgCl2, and 10 mM AIR for 18 h. Unfortunately, the ATP analogue hydrolyzed to ADP. We tried substituting AMPPCP for AMPPNP, but the crystals did not diffract well. A low-resolution structure demonstrated that the AMPPCP bound in a nonproductive manner relative to that observed for ATP in the enzyme 3 Mg2ATP complex. Hence, the ternary complex reported here is that of the enzyme with MgADP and AIR. Relevant X-ray data collection statistics for both protein complexes are listed in Table 1. The structure of N5-CAIR synthetase in complex with Mg2ATP was solved by molecular replacement with the program Phaser (19) using as a search model the structure of the apo form of Candida albicans N5-CAIR synthetase determined in this laboratory (unpublished results). The initial electron density map was subsequently improved by 4-fold averaging with the software package DM (20). An initial model was built using COOT (21). Least-squares refinement with TNT (22) and manual adjustment with COOT reduced the R-factor to 19.8% for all measured X-ray data from 30 to 2.1 A˚ resolution. The coordinates for this structure served as the starting model for refinement of the enzyme 3 MgADP 3 AIR complex. Least-squares refinement of the enzyme 3 MgADP 3 AIR complex converged to 20.6% for all measured X-ray data from 30 to 2.0 A˚ resolution. Relevant refinement statistics are listed in Table 2. Kinetic Analyses of Wild-Type and Mutant Enzymes. The steady-state kinetic parameters for the wild-type and mutant
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Table 2: Least-Squares Refinement Statistics enzyme complexed enzyme complexed with Mg2ATP with MgADP and AIR resolution limits (A˚) 30.0-2.1 30.0-2.0 R-factora (overall) (%)/no. 19.8 (104194) 20.6 (118549) of reflections R-factor (working) (%)/no. 19.6 (93863) 20.3 (106822) of reflections R-factor (free) (%)/no. 26.1 (10331) 26.9 (11667) of reflections no. of protein atoms 11869b 11802c d no. of heteroatoms 981 1345e average B values protein atoms (A˚2) 32.8 32.7 30.8 30.0 ligands (A˚2) solvent (A˚2) 35.7 weighted root-mean-square deviations from ideality bond lengths (A˚) 0.012 0.014 bond angles (deg) 2.60 2.83 trigonal planes (A˚) 0.007 0.008 general planes (A˚) 0.012 0.012 torsional angles f (deg) 18.3 18.8 Ramachandran Statistics 89.5, 10.3, 0.2 89.3, 10.5, 0.2 [core, allowed, generously allowed (%)] P P a R-factor = ( |Fo - Fc|/ |Fo|) 100, where Fo is the observed structure factor amplitude and Fc is the calculated structure factor amplitude. bThese include multiple conformations for Asn 37, Cys 67, Gln 96, and Ser 339 in chain A, Gln 62, Glu 171, Glu 236, Asp 246, and Glu 259 in chain B, Arg 61 and Asn 231 in chain C, and Val 9 in chain D. cThese include multiple conformations for Asp 47, Lys 65, Gln 327, Ser 339, and His 365 in chain A, Gln 62, Asp 78, Glu 120, Lys 233, Asp 246, and Glu 259 in chain B, Gln 29, Asp 47, Lys 65, Gln 96, Arg 112, and Glu 236 in chain C, and Ser 4 in chain D. dHeteroatoms include 4 ATP, 8 Mg2þ, and 849 waters. eHeteroatoms include 4 ADP, 4 Mg2þ, 4 AIR, 1 Naþ, 2 CHES, and 1130 waters. fThe torsional angles were not restrained during the refinement.
enzymes were determined using the ATP-coupled assay system as previously described (17). In a 1 mL cuvette, buffer [50 mM HEPES (pH 7.5), 20 mM KCl, and 6.0 mM MgCl2], 0.2 mM NADH, 2.0 mM phosphoenolpyruvate, 1.0 mM NaHCO3, 4 units of pyruvate kinase, and 17 units of lactate dehydrogenase were added. After the cuvette was equilibrated to 37 °C, N5CAIR synthetase was added (the amount varied depending upon the mutant protein) followed by 1.1 mM ATP. Background levels of ATP hydrolysis were measured, and the reaction was initiated by the addition of AIR (concentration varied from 5 to 500 μM). NADH oxidation was monitored at 340 nm, and the concentration of AIR consumed was calculated using the extinction coefficient for NADH (6200 M-1 cm-1). The background ATPase activity was very low (
